OsTH1 is a key player in thiamin biosynthesis in rice

Thiamin is a vital nutrient that acts as a cofactor for several enzymes primarily localized in the mitochondria. These thiamin-dependent enzymes are involved in energy metabolism, nucleic acid biosynthesis, and antioxidant machinery. The enzyme HMP-P kinase/thiamin monophosphate synthase (TH1) holds a key position in thiamin biosynthesis, being responsible for the phosphorylation of HMP-P into HMP-PP and for the condensation of HMP-PP and HET-P to form TMP. Through mathematical kinetic model, we have identified TH1 as a critical player for thiamin biofortification in rice. We further focused on the functional characterization of OsTH1. Sequence and gene expression analysis, along with phylogenetic studies, provided insights into OsTH1 bifunctional features and evolution. The indispensable role of OsTH1 in thiamin biosynthesis was validated by heterologous expression of OsTH1 and successful complementation of yeast knock-out mutants impaired in thiamin production. We also proved that the sole OsTH1 overexpression in rice callus significantly improves B1 concentration, resulting in 50% increase in thiamin accumulation. Our study underscores the critical role of OsTH1 in thiamin biosynthesis, shedding light on its bifunctional nature and evolutionary significance. The significant enhancement of thiamin accumulation in rice callus upon OsTH1 overexpression constitutes evidence of its potential application in biofortification strategies.


Import of HET
→ HET_c

Supporting table S5.
Primers used in the study.

Sequence 5 ' 3 GATCCATTGAATTGAATTGAAATCFigure S1 .
Figure S1.Solution to the FBA optimization problem maximization of TH1B flux.The objective function of the model was considered the maximization of thiamin monophosphate synthase (TH1B) flux.The correlation of each enzyme activity with the objective flux was carried out by setting the flux of each reaction as values from 0% to 200% of enzyme activity, with increments of 10%.Flux balance analysis (FBA) was run to simulate the optimal flux in TH1B.Reactions that had linear correlation with fluctuations in the objective flux were considered rate-limiting.a) Correlation between TH1 activity and TH1B flux.No correlation was observed.b) Correlation between TH2 activity and TH1B flux.No correlation was observed.c) Correlation between THIC activity and TH1B flux.THIC activity directly correlates with TH1B flux, pointing it as the limiting step of the pathway.d) Correlation between TTPK activity and TH1B flux.No correlation was observed.

Table S2 .
Rate law (RL) equations, kinetic parameters, and respective references for each reaction of included in the kinetic model.  +  ×   +  ×   +  ×  Hydroxyethylthiazole kinase.In cases where the kinetic parameters were not available in the literature, the stoichiometric model of vitamin B1 metabolism was used to predict the reaction flux.

table S4 .
Stoichiometric matrix used in the development of the stoichiometric model.